Hemopexin (HPX) is the first line of defense against heme-mediated damage to cells in hemolytic states. It is also a physiologically-relevant system of heme transport in mammalian cells. The broad long term objectives are to understand the mechanism of heme uptake by HPX; to define the role of the HPXR(s) in heme uptake; to establish whether a heme transporter is closely associated with the HPXR; to determine when and how signals that are triggered by the HPXR lead to gene regulation, particularly that of the protective proteins HO-1 and MT-1; and to establish whether copper plays in heme uptake and signaling in the HPX system; and, if so, to define the copper-protein(s) involved. The specific aims are: 1). To characterize the process of high affinity, specific heme transport from 55Fe-heme-HPX. We will assess the relative contributions of endocytosis, structural features of the heme molecule and reduction of heme in heme-HPX complexes at the cell surface for heme transport. Toward the goal that copper is required for the process of heme uptake needed for heme oxygenase-1 regulation, we will determine whether the copper chelator bathocuproine disulfonate inhibits heme uptake 2). To clone the HPXR using one of four strategies devised and 3). To investigate whether activation of intracellular signaling cascades by the HPXR, triggered by ligand binding, occurs before endocytosis of the HPXR. Cobalt-protoporphyrin IX(CoPP)-HPX is a HPXR ligand that activates signaling cascades without tetrapyrrole transport. We will use biochemical and morphological techniques to determine whether CoPP-HPX stays at the cell surface or undergoes endocytosis like heme-HPX. The regulatory consequences triggered by the HPXR will be compared in wild type and endocytosis-defective cells. The significance of this research is that it will provide fundamental information on the biochemistry of heme uptake as well as activation of signaling cascades and gene regulation (e.g. of ho-1 that accompanies heme uptake) by the HPXR. These are important intrinsically but also because they aid our understanding of heme-related diseases. These range from hemolysis, trauma and ischemia reperfusion injury but also neuro-degenerative conditions, such as stroke. Novel roles for redox-active, nutrient metals in signaling and gene regulation are also likely outcomes.